The balance between the synthesis and the degradation processes of proteins is essential for the maintenance of the cellular homeostasis. Cells have two main metabolic pathways of protein degradation. A large number of the proteins is either digested by proteolytic enzymes in lysosomes or via the ubiquitin-proteasome-system. An imbalance between the protein synthesis and the degradation processes of proteins leads to a series of pathological processes (1).
The 26S proteasomes are protease-complexes that are composed of multiple subunits, perform the ATP-dependent degradation of poly-ubiquitinylated proteins. They are responsible for the majority of the non-lysosomal proteolysis in eukaryotic cells. They consist of the proteolytic 20S proteasome-core particles and carry a lid on one or both ends that is formed out of the regulatory 19S cap-particles (2, 3). The 20S core particle is a cylindrical assembly of 28 subunits that are arranged in 4 stapled heptamer-rings. 2 rings are formed by 7 subunits of the α-type and 2 rings by 7 subunits of the β-type (4, 5). Both inner β-rings form the central area of the cylinder and carry the proteolytic centres. In contrast to the prokaryotic 20S proteasomes that consist of 14 identical alpha and 14 identical proteolytically active subunits of the β-type, eukaryotic 20S proteasomes have only 3 proteolytically active subunits per β-ring. Proteasomes belong to the family of the N-terminal nucleophilic hydrolases (6, 7). A stimulation of mammalian cells with γ-interferon causes the exchange of the 3 active β-subunits β1, β2 and β5 by the immune homologues β1i, β2i, and β5i, leading to the formation of the immunproteasomes, which generate modified cleavage pattern of substrate peptides. It was shown that the functional integrity of the proteasome is essential for a multitude of cellular functions, such as, for example, the metabolic adaptation, cellular differentiation, cell cycle-control, stress response, the degradation of abnormal proteins and the generation of epitopes that are presented through MHC class I-receptors (for a review: see (8, 9)). Proteasomes are an important but not exclusive producer of the antigenic peptides (10, 11).
The dysregulation of the metabolic pathway of the ubiquitin-proteasome-protein degradation causes several diseases in the human, such as, for example, cancer, neurodegenerative, autoimmune- and metabolic diseases. The inhibition of the proteasomes influences the stability of many proteins, such as those that are involved in the regulation of the cell cycle. Thus, selective inhibitors of the multicatalytic proteasomal subunits are attractive targets in the development of drugs (12).
Most of the cells that are treated with proteasomal inhibitors are sensitized for the apoptosis (13, 14). Interestingly, tumour cells are usually are more sensitive against proteasomal inhibition than normal cells. Healthy cells are subject to an arrest of the cell cycle when treated with proteasomal inhibitors, but, nevertheless, in contrast to tumour cells are less prone for apoptosis (15, 16).
Until today, different proteasomal inhibitors were characterised (see FIG. 1). A distinction is made between selective inhibitors (4 lactacystin, 5 TMC-95A, 6 epoxomicin) and non-selective inhibitors (1 dichlorovinylester, 3 MG132) (17).
The most important proteasomal inhibitor is compound 2, also Bortezomib® or VELCADE™ (see FIG. 1). Bortezomib® was registered by the U.S. Food and Drug Administration (FDA) as drug only available on prescription for the treatment of multiple myeloma (18-20).
Another important proteasomal inhibitor is MG132 (compound 3 in FIG. 1). A decisive disadvantage of MG132 is its lack of/low selectivity in the inhibition of proteasomes (1, 17, 22, 38).
Furthermore, WO 96/13266 describes peptidic boric acid and -ester-compounds that are suitable as inhibitors of the proteasomal function.
The proteasomal amide hydrolysis differs from the amide hydrolysis of all other classes of proteases. Thereby, the particular features are the N-terminal threoninees. The mechanism is depicted in FIG. 2. When analysing the crystal structure of the 20S proteasome, it was revealed that Thr1Oγ functions as the nucleophile, and the N-terminal amino group as the acyl-carrier (6). Covalent inhibitors can bind in the active centre, and in particular either via the hydroxyl group of the Thr1Oγ or simultaneously via the free N-terminus and the Thr1Oγ (for a review: see 17).
Effective in vivo inhibitors of the 20S proteasome thus require a high selectivity and at the same time a good ability to penetrate the cellular membranes. Furthermore, they can be characterized in that they covalently bind to the N-terminal threonine.
It is therefore the object of the present invention, to develop improved inhibitors of the proteasome that are characterized in particular by their selectivity to the proteasome as well as their irreversibility, and that are able to penetrate cellular membranes.
According to the invention, this object is solved by providing compounds having the formula
                wherein R1 to R5 and X are selected independently from one another, and wherein        R1 is Boc, Z, Ac or H,        Z is benzyloxycarbonyl,        L is Leu,        X is Leu or Asp(OR4),        R2 is CH2—CH(CH3)2,        R3 is CH2—OH, CH═O, CH(OH)—C≡C-phenyl, CH(OH)—C(O)—NH—R5 or C(O)—C(O)—NH—R5,        R4 is t-butyl, benzyl or H,        R5 is benzyl, 3-picolyl or phenyl,        and pharmaceutically acceptable salts thereof.        
Excluded shall be a compound wherein, if X is Leu, R3 is CH═O, preferably wherein, if X is Leu, R2 is CH2—CH(CH3)2 and R3 is CH═O, particularly preferred wherein, if R1 is Z and X is Leu, R2 is CH2—CH(CH3)2 and R3 is CH═O.
In a preferred embodiment thereof, the invention comprises compounds, wherein                R1 is Boc or Z,        L is Leu,        X is Asp(OR4),        R2 is CH2—CH(CH3)2,        R3 is CH2—OH,        R4 is t-butyl.        
In a further preferred embodiment thereof, the invention comprises compounds, wherein                R1 is Boc, Z or Ac,        L is Leu,        X is Asp(OR4),        R2 is CH2—CH(CH3)2,        R3 is CH═O,        R4 is t-butyl or benzyl.        
In a further preferred embodiment thereof, the invention comprises compounds, wherein                R1 is Z,        L is Leu,        X is Leu,        R2 is CH2—CH(CH3)2,        R3 is C(O)—C(O)—NH—R5,        R5 is benzyl, 3-picolyl or phenyl.        
In a further preferred embodiment thereof, the invention comprises compounds, wherein                R1 is Z,        L is Leu,        X is Leu,        R2 is CH2—CH(CH3)2,        R3 is CH(OH)—C(O)—NH—R5,        R5 is phenyl.        
In a further preferred embodiment thereof, the invention comprises compounds, wherein                R1 is Z,        L is Leu,        X is Leu,        R2 is CH2—CH(CH3)2,        R3 is CH(OH)—C≡C-phenyl.        
Furthermore, compounds are comprised, wherein                R1 is Z,        L is Leu,        X is Leu,        R2 is CH2—CH(CH3)2, phenyl or benzyl        R3 is CH2—O—C(Cl)═C—Cl.        
The invention furthermore provides methods for producing a compound according to the invention. One such method preferably comprises a step of oxidation or reduction. Preferably, the method is characterized in that the oxidation takes place by using hypervalent iodine reagents.
Thereby, the method according to the invention preferably comprises the conversion of amino alcohols into peptide-mimetics (7-12) with a subsequent oxidation into peptide aldehydes (13-18), e.g. by hypervalent iodine reagents. The synthesis is also possible by reducing derivatized amino acid esters into the respective peptide aldehydes.
The synthesis of the compounds 7-18 according to the invention which started from compound 3 (MG132) as a lead-structure, was performed based on the established substrate-preferences of β-secretase (23) by means of standard methods. The synthesis is depicted in scheme I (see also example 1). The condensation of commercially available, protected dipeptides and amino acids with commercially available amino alcohols was followed by the oxidation into the aldehydes by IBX in DMSO (scheme I).

The intermediate, i.e. the alcohol-derivates 7-12, and the tripeptide aldehydes 13-18 were tested for their ability to inhibit the enzyme. The inhibition of the β-secretase was rather slightly pronounced (IC50>200 μM, results not shown), nevertheless, several compounds were found as potent inhibitors of the 20S proteasome.
In general, peptide aldehydes exhibited no selectivity in the inhibition of enzymes. Thus, different groups were tested for their ability to inhibit threonine-proteases.
The non-selective dichlorovinylester 1 (see FIG. 1), which readily reacts with all possible nucleophiles, such as, for example, cysteine, serine and finally also threonine, served as a further lead-structure for the syntheses of compounds according to the invention. In addition, the aim was pursued to reduce the inherent over-activation of this compound. The “removal” of the acyl group of 1 could reduce the non-specific hydrolysis through ubiquitary nucleophiles, and results in quite stable dichlorovinylethers (28). The resulting ethers, the compounds according to the invention 19-20 (for the synthesis see scheme II and example 1), tolerate an acidic environment, but are hydrolysed readily at pH 11 and converted into α-chloroacetates, which, in turn, react with nucleophiles. This dual reactivity which is provided in a cascade-like reaction, corresponds to the specific requirements for an N-terminal threonine-protease-inhibitor.

An analogous dual reactivity can be observed in propargyl-ketones. A similar compound was synthesized, but unfortunately the alcohol 21 (scheme III) withstood the oxidation into the desired ketone.

Thus, the further focus was laid on transition-state-mimetics and inhibitors. Lead-structures, such as statines (38), α-ketoamides, and chloromethyl ketones are well established in the inhibition of proteases. The combination of these structures with a β-selective tripeptide lead to the compounds 22-28 according to the invention (structures of 22-28, see FIG. 3). Compound 22 was prepared from commercial Z-Leu-Leu and chloromethyl leucine (scheme IV and example 1).

The compounds according to the invention 23-25 were obtained through a Passerini-reaction of MG132 (3) with three isonitriles. The subsequent oxidation through IBX in DMSO delivered the α-ketoamides 26-28 (scheme V and example 1).

Proteasomes are involved in a series of different cellular processes. They are important for the control of the cellular cycle and protect cells against apoptosis by maintaining the balance of anti-apoptotic and pro-apoptotic proteins (9, 31, 32). The interest in potent and specific inhibitors that can be used as potential agents against cancer or neoplastic growth, is very high.
The present invention reports on the synthesis of inhibitors that are based on the proteasomal peptide-inhibitor MG132, which is a potent, but non-specific inhibitor. Side-chain modifications of this tripeptide should lead to a higher potency, selectivity and position-specific inhibition of the 20S proteasome. This assumption is based on a series of known and potent peptidic inhibitors (17, 33, 34, 35).
All compounds according to the invention were tested in cell-lysates for their inhibitory capacity. Thus, during the tests with the mimetics as synthesized, the serine-, cysteine- and metal-proteases were blocked with the protease-inhibitor-cocktail complete (Roche). The proteolysis of the hydrophobic substrate Suc-LLVY-AMC was reduced by 10 of the compounds according to the invention as examined (see also example 5, FIG. 4).
The specific inhibition of a single catalytic site is of specific interest for the development of drugs. Thus, the inhibition of the different proteasomal activities of the proteasome was analysed (see also example 6). The different cleavage-preferences of the proteasome were determined by the specific substrate for the hydrophobic (chymotrypsin-like), the trypsine-like and the caspase-like activities of isolated proteasomes. 12 of 22 derivatives according to the invention inhibited proteasomal activities with IC50-values below 10 μM (see table 1). The peptidic derivatives 13 and 15 inhibited all of the proteasomal hydrolytic activities, whereas four compounds (18, 25, 26 and 27) inhibited the chymotryptic and the caspase-like sites.
Nevertheless, one additional aim of this analysis was the identification of completely selective inhibitors of the proteasomal activity. The tripeptidic alcohol 7 (and compound 8 with lower potency) specifically reduced the trypsine-like activity, and the compounds 16, 21, 22 and 28 resulted in an exclusive reduction of the chymotryptic activity. Notably the most potent of the new inhibitors have IC50-values of below 100 nM (7, 15, 28). These are found in the range of the new proteasomal inhibitors that are currently in clinical phases (33).
Notably, the tetrapeptide-inhibitor PSI (Z-Ile-Glu(OtBu)Ala-Leu-CHO) (36) is structurally related with the compound according to the invention 15 (Z-Leu-Asp(OtBu)Leu-CHO), which belongs to the strongest inhibitors (IC50 below 60 nM).
Furthermore, 15 exhibited a low toxicity and was able to penetrate cellular membranes.
The comparison of the inhibitors according to the invention showed that the ligand-side chains provide the main contributions to the specific and fixed interactions with the different proteolytic catalytic centres (FIG. 8 and example 10). Similar observations were made for the alcohol-derivatives, out of which compound 7 is more effective than the other six. Furthermore, very potent inhibitors were identified in the form of chloromethyl ketone (compound 22) and compounds 25-28.
Tumour cells with their accelerated neoplastic growth are often more sensitive against proteasomal inhibitors, compared with normal cells. The clinically tested proteasomal inhibitor Bortezomib® caused growth arrest and apoptosis in sensitive tumour cells, whereas “normal” cells tolerate higher inhibitor cells (37). The restriction to myeloma tumours could be overcome by more specific inhibitors, such as PSI, which blocked angiogenesis and thus modulated the growth of solid tumours (36). The differences in cellular properties and the predictable resistance-mechanisms required a continuous development of novel proteasomal inhibitors. Efficient cell-permeation, stability in aqueous systems, and the potent induction of cellular events are all obligatory for clinical uses.
Thus, the ability to permeate of the compounds according to the invention and the in vivo influence on proteasomes was tested, and an accumulation of poly-ubiquitinylated proteins in cultivated cells was observed. A more 50% reduction of the intracellular proteasomal activity was observed for 5 of the inhibitors (15, 22, 25, 26, 28) already after 20 hours of incubation. Notably, the proteasomal activity was reduced to 10% in the presence of 15, 26 and 28. Weak inhibitors have a lower influence on the cellular function. The results of the present invention show potency, membrane-permeation and sufficient stability during the incubation periods for the inhibitors 15, 22, 25, 26 and 28. The cellular proteasomal activity is unambiguously reduced and is accompanied by a strong induction of apoptosis following 20-hour treatment with 1 μM of the inhibitors (15, 26, 28). The known increased sensitivity of tumour cells against proteasomal inhibition was confirmed for inhibitor 15 and 28. Surprisingly, a strong induction of the apoptosis was observed in cells that were pre-incubated with compound 7, which inhibited the trypsin-like activity in an exclusive manner. These results indicate that the trypsin-like activity is of particular importance for anti-apoptotic processes.
According to the invention the compounds can be used for the induction of apoptosis in cells.
Furthermore, the compounds according to the invention can be used for the inhibition of the proteolytic activity of the 20S proteasome, 26S proteasome and/or immunoproteasome. Therein they are used for the in vitro, in vivo and/or intracellular inhibition.
Preferably, thereby specifically the trypsin-like activity of the 20S proteasome and 26S proteasome and/or immunoproteasome is inhibited.
Further preferred specifically the chymotrypsin-like activity of the 20S proteasome and 26S proteasome and/or immunoproteasome is inhibited.
Nevertheless, preferably also the chymotrypsin-like, trypsin-like and caspase-like activities of the 20S proteasome and 26S proteasome and/or immunoproteasome can be simultaneously inhibited.
It is furthermore preferred according to the present invention to use the compounds for the treatment of diseases, such as for the treatment of the following therapeutic fields:
Neurology
Inhibitions or malfunctions of proteasomes are associated with the development of Alzheimer's disease, Parkinson's disease, and the Pick-disease. Proteasomes are involved in amyotrophic lateral sclerosis (ALS), in diseases of motor neurons, the polyglutamine-disease and muscular dystrophies.
Tumour Diseases
Proteasomes play a role in the malign transformation, regulation of the cell cycle, inhibition or execution of apoptosis, respectively, degradation of several tumour suppressor-products (APC, p53, Rb), degradation of proto-oncogenes (Raf, Myc, Myb, Rel, Src etc.), malfunctions in the cell cycle regulation. Proteasomes are responsible for the degradation of cyclines, CDK's and inhibitors thereof; an inhibition of proteasomes in most cases leads to an arrest of the cycle.
Viral Diseases
The presentation of viral antigens requires their generation through proteasomes: e.g. HCMV, hepatitis (HCV and HBV), herpes (HVP) and others. In addition, for coxsackie (CVB) and HCMV a role of proteasomes in the viral replication is likely (39, 40)
Endocrinology
Glucocorticoids upregulate, for example, a proteasomal alpha-subunit. The degradation of proteins during the thyroxin-formation takes place through proteasomes.
Immunology
There is an involvement of proteasomes in inflammatory reactions (MHC class I ligands, induction of immunoproteasomes through cytokines). Furthermore, a possible role in the generation and the progression of autoimmune diseases exists. A determination of proteasome-antibodies is possible in the serum of SLE-, Sjögren syndrome- and polymyositis-patients, partially a detection of circulating, released proteasome in the serum of these patients is possible (41).
The present invention furthermore provides pharmaceutical compositions, comprising one or more of the compounds according to the invention or a pharmaceutically acceptable salt thereof together with pharmaceutically acceptable carriers and/or excipients. Such pharmaceutically acceptable carriers and excipients are known to the person of skill.
The pharmaceutical compositions according to the invention are characterized in that the compound(s) are present in an amount that a concentration range of preferably 0.001 to 100 μM, further preferred of 0.01 to 10 μM at the treatment in vivo.
They are furthermore characterized in that the compound(s) are present in an amount which effectively inhibits the proteasome-function in a cell or a mammal.
The present invention furthermore provides a method for the inhibition of the growth of a cancer cell, comprising contacting of a cell with a compound according to the invention or with a pharmaceutical composition according to the invention.
The present invention shall now be illustrated by the following examples with reference to the accompanying Figures, nevertheless, without being limited to the examples.